The present invention relates to oligonucleotides having a novel sugar-phosphate backbone containing internucleoside 3xe2x80x2-NHP(O)(Sxe2x88x92)O-5xe2x80x2 linkages. More particularly, the present invention is directed to thiophosphoramidate oligonucleotide compositions, their use as diagnostic or therapeutic agents and methods for synthesizing thiophosphoramidate oligonucleotides.
Nucleic acid polymer chemistry has played a crucial role in many developing technologies in the pharmaceutical, diagnostic, and analytical fields, and more particularly in the subfields of antisense and anti-gene therapeutics, combinatorial chemistry, branched DNA signal amplification, and array-based DNA diagnostics and analysis (e.g. Uhlmann and Peyman, Chemical Reviews, 90:543-584, 1990; Milligan et al., J. Med. Chem., 36:1923-1937, 1993; DeMesmaeker et al., Current Opinion in Structural Biology, 5:343-355, 1995; Roush, Science, 276:1192-1193, 1997; Thuong et al., Angew. Chem. Int. Ed. Engl., 32:666-690, 1993; Brenner et al., Proc. Natl. Acad. Sci., 89:5381-5383, 1992; Gold et al., Ann. Rev. Biochem., 64:763-797, 1995; Gallop et al., J. Med. Chem., 37:1233-1258, 1994; Gordon et al., J. Med. Chem., 37:1385-1401, 1994; Gryaznov, International application PCT/US94/07557; Urdea et al., U.S. Pat. No. 5,124,246; Southern et al., Genomics, 13: 1008-1017, 1992; McGall et al., U.S. Pat. No. 5,412,087; Fodor et al., U.S. Pat. No. 5,424,186; Pirrung et al., U.S. Pat. No. 5,405,783).
Much of this chemistry has been directed to improving the binding strength, specificity, and nuclease resistance of natural nucleic acid polymers, such as DNA. Unfortunately, improvements in one property, such as nuclease resistance, often involve trade-offs against other properties, such as binding strength. Examples of such trade-offs abound: peptide nucleic acids (PNAs) display good nuclease resistance and binding strength, but have reduced cellular uptake in test cultures (e.g. Hanvey et al., Science, 258:1481-1485, 1992); phosphorothioates display good nuclease resistance and solubility, but are typically synthesized as P-chiral mixtures and display several sequence-non-specific biological effects (e.g. Stein et al., Science, 261:1004-1012, 1993); methylphosphonates display good nuclease resistance and cellular uptake, but are also typically synthesized as P-chiral mixtures and have reduced duplex stability (e.g. Mesmaeker et al. (cited above); and so on.
Recently, a new class of oligonucleotide analog has been developed having so-called N3xe2x80x2xe2x86x92P5xe2x80x2 phosphoramidate internucleoside linkages which display favorable binding properties, nuclease resistance, and solubility (Gryaznov and Letsinger, Nucleic Acids Research, 20:3403-3409, 1992; Chen et al., Nucleic Acids Research, 23:2661-2668, 1995; Gryaznov et al., Proc. Natl. Acad. Sci., 92:5798-5802, 1995; and Gryaznov et al., J. Am. Chem. Soc., 116:3143-3144, 1994). Phosphoramidate compounds contain a 3xe2x80x2-amino group at each of the 2xe2x80x2-deoxyfuranose nucleoside residues replacing a 3xe2x80x2-oxygen atom. The synthesis and properties of oligonucleotide N3xe2x80x2xe2x86x92P5xe2x80x2 phosphoramidates are also described in Gryaznov et al., U.S. Pat. Nos. 5,591,607; 5,599,922; 5,726,297; and Hirschbein et al., U.S. Pat. No. 5,824,793.
The oligonucleotide N3xe2x80x2xe2x86x92P5xe2x80x2 phosphoramidates form unusually stable duplexes with complementary DNA and especially RNA strands, as well as stable triplexes with DNA duplexes, and they are also resistant to nucleases (Chen et al., Nucleic Acids Research, 23:2661-2668, 1995; Gryaznov et al., Proc. Natl. Acad. Sci., 92:5798-5802, 1995). Moreover oligonucleotide N3xe2x80x2xe2x86x92P5xe2x80x2 phosphoramidates are more potent antisense agents than phosphorothioate derivatives both in vitro and in vivo (Skorski et al., Proc. Natl. Acad. Sci., 94:3966-3971, 1997). At the same time the phosphoramidates apparently have a low affinity to the intra- and extracellular proteins and increased acid liability relative to the natural phosphodiester counterparts (Gryaznov et al., Nucleic Acids Research, 24:1508-1514, 1996). These features of the oligonucleotide phosphoramidates potentially adversely affect their pharmacological properties for some applications. In particular, the acid stability of an oligonucleotide is an important quality given the desire to use oligonucleotide agents as oral therapeutics.
In order to circumvent the above described problems associated with oligonucleotide analogs, a new class of compounds was sought that embodies the best characteristics from both oligonucleotide phosphoramidates and phosphorothioates. The present invention describes the synthesis, properties and uses of oligonucleotide N3xe2x80x2xe2x86x92P5xe2x80x2 thiophosphoramidates.
The compositions and methods of the present invention relate to polynucleotides having contiguous nucleoside subunits joined by intersubunit linkages. In the polynucleotides of the present invention, at least two contiguous subunits are joined by a N3xe2x80x2xe2x86x92P5xe2x80x2 thiophosphoramidate intersubunit linkage defined by the formula of 3xe2x80x2-[xe2x80x94NHxe2x80x94P(xe2x95x90O)(xe2x80x94SR)xe2x80x94Oxe2x80x94]-5xe2x80x2, wherein R is selected from the group consisting of hydrogen, alkyl, aryl and salts thereof. In a preferred embodiment of the invention, R is hydrogen or a salt thereof. The inventive polynucleotides can be composed such that all of the intersubunit linkages are N3xe2x80x2xe2x86x92P5xe2x80x2 thiophosphoramidate. Alternatively, the polynucleotides of the invention can contain a second class of intersubunit linkages such as phosphodiester, phosphotriester, methylphosphonate, Pxe2x80x23xe2x86x92N5xe2x80x2 phosphoramidate, Nxe2x80x23xe2x86x92P5xe2x80x2 phosphoramidate, and phosphorothioate linkages.
An exemplary N3xe2x80x2xe2x86x92P5xe2x80x2 thiophosphoramidate intersubunit linkage has the formula: 
where B is a purine or pyrimidine or an analog thereof, Z is OR, SR, or methyl, wherein R is selected from the group consisting of hydrogen, alkyl, and aryl and their salts; and R1 is selected from the group consisting of hydrogen, Oxe2x80x94R2, Sxe2x80x94R2, and halogen, wherein R2 is H, alkyl, or (CH2)nW(CH2)mH, where n is between 1-10, m is between 0-10 and W is O, S, or NH, with the proviso that when Z is methyl or OMe, R1 is not H. The nucleoside subunits making up the polynucleotides can be selected to be in a defined sequence: such as, a sequence of bases complementary to a single-strand nucleic acid target sequence or a sequence that will allow formation of a triplex structure between the polynucleotide and a target duplex. The nucleoside subunits joined by at least one N3xe2x80x2xe2x86x92P5xe2x80x2 thiophosphoramidate intersubunit linkage, as described above, have superior resistance to acid hydrolysis, yet retain the same thermal stability as compared to oligonucleotides having phosphoramidate intersubunit linkages.
The present invention also includes a method of synthesizing an oligonucleotide N3xe2x80x2xe2x86x92P5xe2x80x2 thiophosphoramidate. In this method a first nucleoside 5xe2x80x2-succinyl-3xe2x80x2-aminotrityl-2xe2x80x2,3xe2x80x2-dideoxy nucleoside is attached to a solid phase support. The first nucleoside additionally has a protected 3xe2x80x2 amino group. The protected 3xe2x80x2 amino group is then deprotected to form a free 3xe2x80x2 amino group to which a second nucleoside is added. The free 3xe2x80x2 amino group of the first nucleoside is reacted with a 3xe2x80x2-protected aminonucleoside-5xe2x80x2-O-cyanoethyl-N,N-diisopropylaminophosphoramidite monomer to form an internucleoside N3xe2x80x2xe2x86x92P5xe2x80x2 phosphoramidite linkage. The internucleaside phosphoramidite group is then sulfurized to form a N3xe2x80x2xe2x86x92P5xe2x80x2 thiophosphoramidate internucleaside linkage between the first and second nucleosides.
In another embodiment of the invention, a method is provided for hybridizing a thiophosphoramidate oligonucleotide of the invention to a DNA or RNA target. The thiophosphoramidate polynucleotide comprises a sequence of nucleoside subunits joined by at least one subunit defined by the formula: 
where B is a purine or pyrimidine or an analog thereof, Z is OR, SR, or methyl, and R1 is selected from the group consisting of hydrogen, Oxe2x80x94R2, Sxe2x80x94R2, and halogen, wherein R2 is H, alkyl, or (CH2)nW(CH2)mH, where n is between 1-10, m is between 0-10 and W is O, S, or NH, with the proviso that when Z is methyl or OMe, R1 is not H. The thiophosphoramidate polynucleotide is contacted with the RNA target to allow formation of a hybridization complex between the polynucleotide and the RNA target.
The present invention also includes pharmaceutical compositions and kits including a polynucleotide having at least one N3xe2x80x2xe2x86x92P5xe2x80x2 thiophosphoramidate linkage, as described above. The inventive oligonucleotides are particularly useful in oral therapeutic applications based on hybridization, such as, antigene and antisense applications, including the inhibition of telomerase enzyme activity.